Efficient inhibition of hepatitis B virus replication by hepatitis delta virus ribozymes delivered by targeting retrovirus

Abstract

Background: Hepatitis delta virus (HDV) ribozyme is an attractive molecular tool that can specifically recognize and catalyze the self-cleavage of the viral RNA phosphodiester backbone. However, a major obstacle in the medical application of the HDV ribozyme is the lack of specificity in the delivery of the ribozyme to defined target cells.

Results: The objective of this study was to determine whether retroviral vectors can deliver the HDV ribozyme into the target cells and to elucidate whether HDV ribozyme plays a role in hepatitis B virus (HBV) replication. In our study, the transduction of helper-free pseudotyped retrovirus, which showed a broad host range, in human hepatoma cells was performed under 2 conditions, that is, in the presence of polymerized human serum albumin (pHSA) and in the absence of pHSA. The transduction ability in the presence of pHSA was higher than in the absence of pHSA. Moreover, HBsAg and HBeAg levels after transductions with pHSA were significantly lower than those in the absence of pHSA, thus indicating that the recombinant retrovirus had HBV-specific cleavage activity and targeted HepG2215 cells.

Conclusions: These data suggest that this system provides a new approach for targeting hepatocytes and has a great potential in gene therapy for HBV infection.

Figure 1

Inhibition of HBsAg and HBeAg secretion in HepG2215 cell supernatants by HDV ribozyme with or without pHSA. A: HepG2215 cells were transduced with a retrovirus carrying the HDV ribozyme with and without pHSA. At 48 hours after infection, the supernatants were collected, and ELISA was performed to detect HBsAg. The level of HBsAg was significantly decreased with pHSA (P < 0.001). The values are the mean ± S.D. values of 3 independent tests; B: ELISA detecton of HBeAg. Rz could significantly inhibit HBeAg expression with pHSA (P = 0.001).

Figure 2

HBV DNA copies in HepG2215 cells. HBV DNA was extracted and real-time PCR was performed (p < 0.001).

Figure 3

Construction of expression plasmids. The expression plasmid for the chimeric envelope was assembled by cloning HBV preS2 and ENV coding sequences to multiple cloning sites of the eukaryotic expression plasmid pcDNA3.1(-). The insertion sites are Hind III and XhoI in mutated ENV. The pGAG-POL and pRz recons expressed Moloney murine leukemia virus (Mo-MLV), Gag-Pol protein, and HDV ribozyme, respectively. The ENV and GAG-POL expression plasmid contains an MoMLV CMV-driven promoter, while Rz is an MoMLV LTR promoter. All plasmids contain SV40 origins of replication. SP, signal peptide.